Download Multi-copy suppressor screen

Yeast transformation
Genetic manipulation of yeast is not limited to mating and sporulation. Yeast will take up
DNA if cells are treated the right way. This process is called transformation.
Of the many cells that are treated, only a few cells actually take up the DNA. Thus
transformation is a rare event. However rare events can be selected for. A part of the
procedure involves selecting for the cells that are transformed, that is cells that have
taken up DNA and are expressing the genes present on that piece of DNA.
In the simple demonstration experiment today, you are using a recipient cell that is ura3-,
and a DNA molecule that carries a wild type copy of the URA3 gene. To select for
transformants, you will plate the cells on YMD-uracil media, so that only recipients that
take up and express URA3 can grow.
To prove that the cells that grow are transformants and not revertants of the ura3mutation in the recipients, it is necessary to perform a “no DNA control”. This is done by
performing a mock transformation procedure on cells, without adding any DNA, and
plating them on a YMD-uracil plate.
DNA can be introduced into yeast in a number of formats and has a number of possible
fates, depending on the kinds of sequences present on the DNA and whether and where
the DNA is linearized. In the experiment today, the DNA you are using is a circular,
supercoiled DNA isolated from E. coli as a plasmid. Because these plasmids (for example
pRS316) have an ARS (autonomously replicating sequence), copies of the DNA will be
produced once the plasmid enters the yeast cell. This plasmid also has a centromere that
allows the plasmid to segregate during mitosis. Thus this plasmid is in essence a
miniature chromosome, once it enters the yeast nucleus. These centromeric plasmids are
present as a single copy in the cell.
DNA that has no ARS will not be maintained in the yeast cell because it cannot be
duplicated (during replication). To enable a piece of DNA lacking an ARS to replicate,
you need to insert it into the yeast chromosome where it will be replicated using
chromosomal ARS elements adjacent to the site of integration. In yeast recombination is
almost always homologous, so that the transformed DNA must have sequences that
match the chromosome almost exactly in order to recombine and insert into the
chromosome. Also, free DNA ends in yeast are extremely “recombinogenic”, that is the
recombination machinery assembles on free DNA ends, identifies the homologous
sequences and catalyzes recombination resulting in the insertion of the DNA into the
chromosome. This feature of yeast allows targeted gene insertions as well as gene
disruptions and other sorts of sophisticated genetic engineering.
Yeast also have a natural DNA plasmid called the 2 micron circle, that is present at 30-50
copies per cell. Recombinant plasmids have been created that have convenient selectable
markers and the replication origin of the 2 micron circle. These plasmids also replicate at
30-50 copies per yeast cell. This allows higher levels of expression of genes carried on
such plasmids. A plasmid called pRS326 has the URA3 gene and a polylinker that is the
same as pRS316 but with the 2 micron origin of replication. If foreign genes are present
in 2 micron plasmids, then these genes are present as 30-50 copies in the cell and the
effect of having multiple copies of genes in a cell can be investigated. This is what we
will be investigating in this experiment.
An additional feature of the URA3 locus is that it is “counter-selectable”. This means that
there exists a selection for the loss of URA3 function. A compound called 5-fluoroorotic
acid (5-FOA) is converted to a uracil analog by the enzyme encoded by URA3.
Incorporation of the uracil analog into RNA instead of uracil is poisonous to the cell. If
the URA3 enzyme is absent (and uracil is provided to the cells) 5-FOA is not poisonous.
Thus there is “forward” selection for URA3 on YMD-uracil medium, and “counterselection” on YMD+5-FOA medium.
This is also the basis for plasmid shuffling, a technique that allows mutations of a gene
on a second plasmid to be tested, even though they may be lethal. The trick is to have the
wild type gene on a URA3 plasmid and introduce the mutant gene on a second plasmid
(LEU2 plasmid). Then you see if the strain will grow on medium containing 5-FOA.
Cells will lose the URA3 plasmid when grown on 5-FOA. When they lose the URA3
plasmid carrying the wild type gene, they are only left with the mutant version of the
gene (on the LEU2 plasmid). If the mutant gene is still functional, cells will be able to
grow on 5-FOA, otherwise they will be unable to grow and will die. The mutant gene
might show novel phenotypes caused by the mutation such as temperature sensitivity.
Calculation exercise:
Assume you have 1 ug of plasmid DNA. What is the molecular weight of pRS316?
How many plasmid molecules are present in 1 ug of DNA?
Screening a high copy library for disruptors of silencing.
The natural 2 micron plasmid in yeast is present at 30-50 copies per cell. The origin of
replication from this natural plasmid has been used to create shuttle plasmids with
selectable markers and polylinker cloning sites. Insertion of DNA in these plasmids
allows us to test the effect of overexpressing a gene.
-One effect of over expressing a gene is that increased concentration of a gene product
can bolster the activity of another gene product, which has suffered a mutation.
-Alternatively, over expression may bypass the need for a mutant gene product.
-Finally over expression of a protein may alter the correct stoichiometry leading to
mutant phenotypes. For example, if a protein-A interacts with protein-B in the ratio 2:1
for normal function, then over expression of protein-B may lead to sub-stoichiometric
complexes and loss of normal function.
This phenomenon can be used to search for and clone proteins that function in a
particular complex.
The search makes use of a “high-copy library”, which is a collection of plasmids that
represents the entire genome of yeast, carried in the plasmid pRSXXXX (2micron,
URA3, ampr).
The collection of plasmids present in this library has the following properties:
70% of the plasmids have inserts.
The average size of the insert is 10 kb.
The number of different plasmids is sufficient to contain the equivalent of 10 yeast
genomes (one genome = 13,000 kb).
The number of yeast transformants needed to be 99% sure that each segment of the yeast
genome has been tested for high copy phenotypes is 60,000.
Each team will attempt to create 10,000 transformants. (2000)
Studies of the HMR locus show that a multi protein complex composed of the Sir
proteins- Sir2, Sir3p and Sir4p silences this locus. Studies have also shown that overexpression of these proteins disrupts silencing presumably by disrupting the correct
stoichiometry of the proteins in the complex.
We wish to determine proteins that when over-expressed affect silencing. If we identify
additional proteins that disrupt silencing, then we will attempt to determine if these
proteins interact with the Sir proteins. We want to know if there are proteins in the yeast
genome that support the function of the Sir proteins, who these proteins are, what they do
in silencing. If in fact they do exist, then we will clone them. To test this hypothesis, we
will transform the high copy library into a specific strain and see if any genes disrupt
silencing of the HMR locus. When the locus is silenced the colonies are red and when the
locus is derepressed, the colonies turn white.
Today we will perform a transformation. We’ll use a method that gives high efficiency
transformation.
Each team will do a transformation, plate them on 10 plates and place the plates at 30C
We will test colonies that grow for color development.
Protocol
Grow strain in 10 ml YPD overnight
Next day measure OD 600
Inoculate 100ml YPD with overnight culture so that OD is 0.1/ml (i.e. 10 OD of cells)
Grow till OD = 0.8 at 30C
Spin down cell 3 min 3000 rpm
Resuspend 10 ml 1xTEL and shake overnight at Room temp
Next day spin down cells 3 min 3000 rpm
Resuspend in 1 ml 1xTEL.
Leave 30 min. on the bench at Room temp.
In a eppendorf tube add
Sonicated Salmon Sperm DNA (10 mg/ml)
Library DNA (0.2 ug)
Competent Cells
5 ul
5 ul
100 ul
Mix by pipeting up and down
Incubate room temp without shaking for 30 min.
Add 700 ul 40% PEG/TEL
Mix by pipeting up and down
Incubate room temp without shaking for 60 min.
Heat shock 42C 10 min.
Spin cells gently (setting 7 in Eppendorf microfuge) for 10 sec.
Resuspend cells in 1 ml sterile water and plate on plates selecting for the plasmid. You
want to get about 200 colonies per plate.
Grow 30C for two days.
5xTEL
0.5M LiAc
50 mM Tris HCl pH 7.5
5 mM EDTA
Final volume
Filter sterilize and store RT
50ml of 1M stock
5ml of 1M stock
1ml of 0.5M stock
100ml
1xTEL
Dilute the 5x TEL with sterile water to 1x and use
50% PEG 4000
Dissolve 50g PEG4000 in 100 ml water.
Filter sterilize and store
PEG/TEL
80ml 50% PEG4000
20 ml 5xTEL
Salmon sperm DNA
Dissolve in water to 10 mg/ml
Sonicate 1 min. x3
boil 10 min. and aliquot.
ROY99
1
Transform with no library DNA. Resuspend cells in 200 ul water. Plate on one –
ura 5ug/ml ade HC YMD plate.
2
Transform with 0.2 ug of library DNA. Resuspend the cells in 1000 ul water.
Plate on 5-10 –ura 5ug/ml ade HC YMD plates (100-200 ul per plate)
5-FOA- Plates
Ingredients
5-FOA Media
1
Yeast Nitrogen Base
(without amino acids)
5- FOA (Fluoro orotic acid)
Uracil
Glucose
Water
Heat to 65 C to dissolve. Filter Sterilize
Amount
7g
1g
50 mg
20 g
500 ml
2
Bacto-Agar
Water
Autoclave
20 g
500 ml
Mix solution 1 and 2 thoroughly and pour plates.
Label plates.
Supplement the plates with 300 ul of ALL mix before plating cells.
YMD color Plates
–ura + 5ug/ml ade +HC YMD plates (1 lit)
Yeast nitrogen base (without aa + ammonium sulfate)
Glucose
agar
water
Sterilize by autoclaving
Then after cooling to 65C, add
10x HC stock
100x color development stock of amino acids.
Pour plates
6.7g
20g
20g
890 ml
100 ml
10 ml
10x Hartwell complete stock solution (500ml)
L-methionine
0.1g
L-tyrosine
0.3g
L-isoleucine
0.4g
L-phenylalanine
0.25g
L-glutamic acid
0.5g
L-threonine
1.0g
L-aspartic acid
0.5g
L-valine
0.75g
L-serine
2.0g
L-arginiine
0.1g
Filter sterilize.
100x stock for color development assay: (100 ml)
Adenine
50 mg (5 ug/ml)
Uracil
350 mg
L-lysine
1200 mg
L-tryptophan
800 mg
L-leucine
800 mg
L-Histidine
200 mg
Take white colonies and re-streak on fresh plates –ura + 5ug/ml ade+HC
Then streak on 5-FOA plates. Take colonies from 5-FOA plates and streak on
Streak on +ura+5ug/ml ade+HC and check for color development.
Plasmid Rescue from Yeast
1. Grow 5ml YMD-ura culture overnight.
2. Transfer 1.5ml of cells to an eppendorf tube, pellet cells and resuspend in 100 ul of
STET.
STET
8% sucrose
50 mM Tris pH 8
50 mM EDTA
5% Triton
water
for 100 ml
8g
5.0 ml 1M stock
10 ml 0.5M stock
5 ml of 100%
to 100 ml
3. Add 0.2 g of glass beads and vortex vigorously for 5 min.
4. Add 100 ul STET, vortex briefly and place in boiling water bath for 3 minutes.
5. Cool briefly on ice and spin 10 min at 4°C.
6. Transfer 100 ul of the supernatant to a fresh tube containing 50 ul of 7.5M ammonium
acetate. Incubate at –20°C for 1 hour and then spin for 10 min at 4°C. Note, the time
spent at –20°C is impt., don’t go longer.
7. Add 100 ul of the supernatant to 200 ul ice cold EtOH.
8. Spin 10’ and then wash pellet with 70% EtOH.
9. Resuspend pellet in 20 ul water and use 5-10 ul of this to transform competent bacteria.
(In general, you should use MH1, which give a better transformation efficiency)
Reference: NAR 20(14) 3790.
Bacterial Transformation
1
2
3
4
5
6
put 50 l of competent bacteria into a sterile 1.5 ml eppendorf tube.
add DNA and incubate on ice for 15-20 minutes.
heat shock for 2 mintues at 37o C and add 0.5 ml of warm LB
incubate at 37o C for 45 minutes to 1 hour
pellet cells and resuspended in desired volume
plate onto LB-Amp plates and incubate overnight at 37o C
Plasmid isolation from bacteria (Promega)
Pellet 1-3 ml of E. coli cells by centrifugation for 1-2 minutes at 10,000 rpm in a
microcentrifuge. Pour off the supernatant, blot the tube upside down on a kim wipe to
remove excess media.
Completely resuspend cell pellet in 200 ul of cell resuspension solution.
Add 200 ul of cell lysis solution and mix by inverting the tube 5 times. The cell
suspension should clear immediately.
Add 200 ul of neutralizing solution and mix by inverting the tube 5x times.
Centrifuge the lysate at 10,000 g in a microcentrifuge for 5 min.
Take supernatant.
Prepare one Wizard minicolumn. Remove the plunger from a 3ml disposable syringe and
set it aside. Attach the syringe barrel to the luer lock extension of the minicolumn.
Vortex/mix the resin first. Pipet 1 ml of the resuspended resin into the barrel (if crystals
or aggregates are present, dissolve by warming the resin to 37 C for 10 min. Cool to 30 C
before use.)
Carefully remove the cleared bacterial supernatant from each miniprep and transfer it to
the barrel of the minicolumn/syringe assembly containing the resin.
Carefully insert the syringe plunger and gently push the slurry into the minicolumn.
Detach the syringe from the minicolumn and remove the plunger from the syringe barrel.
Reattach the syringe barrel to the minicolumn.
Pipet 2ml of column wash solution (after the addition of ethanol) into the barrel of the
minicolumn/syringe assembly. Insert the plunger into the syringe and gently push the
column wash solution through the minicolumn.
Remove the syringe and transfer the minicolumn to a 1.5 ml microcentrifuge tube.
Centrifuge the minicolumn at 10,000 g n a microcentrifuge for 2 min to dry the resin.
Transfer the minicolumn to a new 1.5 ml tubes. Add 50ul of nuclease free water to the
minicolumn. Wait 1 minute. Centrifuge at 10,000g in the microcentrifuge for 20sec to
elute the DNA.
Remove and discard the minicolumn. DNA is stable in water at -20C.